Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Cindy Bokobza; Alice Jacquens; Manuela Zinni; Val&#233;rie Faivre; Jennifer Hua; David Guenoun; Caroline Userovici; Shyamala Mani; Vincent Degos; Pierre Gressens; Juliette Van Steenwinckel

doi:10.3791/62964

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

일차 세포 배양을 위한 Neonate 마우스의 미세아교세포의 자기 분리

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07:23 min

July 25, 2022

DOI: 10.3791/62964-v

Cindy Bokobza*¹, Alice Jacquens*^1,2, Manuela Zinni¹, Valérie Faivre¹, Jennifer Hua¹, David Guenoun¹, Caroline Userovici¹, Shyamala Mani¹, Vincent Degos^1,2, Pierre Gressens¹, Juliette Van Steenwinckel¹

¹NeuroDiderot, Inserm UMR-1141, Hôpital Robert Debré 48,Université de Paris, ²Department of anesthesia and critical care,APHP-Sorbonne university

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Overview

This study presents a reproducible protocol for isolating primary microglia from neonatal mice, using magnetic sorting technology. The methodology allows the culture of microglia under conditions that closely replicate in vivo characteristics, providing insights into their phagocytic activity.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Microglia Research

Background

Microglia play crucial roles in the central nervous system.
Understanding microglial responses to stimuli is important for neuroinflammatory research.
Magnetic cell sorting provides a viable strategy for isolating microglia.
Previous protocols may lack reproducibility or relevance to in vivo conditions.

Purpose of Study

To develop a protocol for efficiently isolating and culturing primary microglia.
To allow for the assessment of microglial responses to inflammatory stimuli.
To evaluate the purity and viability of isolated microglia.

Methods Used

Cell culture method utilizing magnetic sorting to isolate microglia from neonatal mice.
The biological model involves cortical microglia from neonate pups.
Immunochemistry and flow cytometry were employed to assess cell purity.
Key steps include dissection, enzymatic dissociation, and sorting using CD11b microbeads.
Phagocytic assays evaluated microglial activity in response to pro-inflammatory factors.

Main Results

Microglia demonstrated increased phagocytic activity in response to specific inflammatory conditions.
Increased cell viability and purity were confirmed through flow cytometry post-sort.
Confocal microscopy highlighted pronounced differences in phagocytic activity based on stimulation duration.
The method clarified distinctions between different brain cell populations.

Conclusions

This study establishes a reliable method for isolating mouse microglia for in vitro experimentation.
The findings enhance the understanding of microglial functions and their responses to inflammation.
This protocol serves as a foundation for further investigations into neuroinflammatory processes.

Frequently Asked Questions

What are the advantages of using this isolation method for microglia?

This method ensures high purity and viability of isolated microglia, closely mimicking in vivo conditions, which enhances experimental relevance.

How are microglia cultured after isolation?

After isolation, microglia are cultured in specialized medium and stimulated with inflammatory factors to assess their functional responses.

What outcomes can be measured using this protocol?

Outcomes include microglial viability, phagocytic activity, and response to inflammatory stimuli assessed via confocal microscopy and flow cytometry.

Can this method be adapted for other types of brain cells?

While this protocol is specifically designed for microglia, adaptations may be possible for other cell types by modifying the sorting antibodies.

What are the limitations of this microglia isolation protocol?

Limitations may include the need for precise dissection and careful handling to avoid mechanical damage to cells during sorting.

What type of analysis can be performed after isolating microglia?

Following isolation, various analyses such as transcriptomic studies, immunochemistry, and functional assays can be conducted to explore microglial roles.

1차 미세아교세포 배양은 일반적으로 새로운 항염증 분자를 평가하는 데 사용됩니다. 본 프로토콜은 신생아 새끼로부터 미세아교세포를 자기적으로 분리하는 재현 가능하고 관련성 있는 방법을 설명합니다.

이 프로토콜을 통해 우리는 생체 내 특성을 밀접하게 모방하는 조건에서 미세아교세포를 배양할 수 있습니다. 자성 세포 분류 기술을 사용하는 당사의 프로토콜을 사용하면 배양 배지에서 혈청을 사용하지 않고 시험관 내에서 이틀만 미세아교세포를 자극할 수 있습니다. 시작하려면 작은 가위를 사용하여 15-20 밀리미터의 시상 봉합사 후 목에서 코까지 피부를 자릅니다.

구멍 매그넘이 두개골과 평행하도록 팁을 삽입하십시오. 양쪽에서 눈으로 자릅니다. 작은 가위로 눈 사이를 잘라 머리에서 두개골과 뇌를 분리하십시오.

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